Molecular Dissection of Bombyx mori Nucleopolyhedrovirus orf8 Gene

Viruses including baculoviruses are obligatory parasites, as their genomes do not encode all the proteins required for replication. Therefore, viruses have evolved to exploit the behavior and the physiology of their hosts and often eoevolved with their hosts over millions of years. Recent comparative analyses of complete genome sequences of baculoviruses revealed the patterns of gene acquisitions and losses that have occurred during baculovirus evolution. In addition, knowledge of virus genes has also provided understanding of the mechanism of baculovirus infection including replication, species-specific virulence and host range. The Bm8 gene of Bombyx mori nucleopolyhedrovirus (NPV) and its homologues are found only in group I NPV genomes. The Autographa californica NPV Acl6 gene is a homologue of Bm8 and, encodes a viral structural protein. It has been shown that Bm8/Ac 16 interacts with baculoviral and cellular proteins. Bm8/Ac 16 interacts with baculoviral IE1 that is facilitated by coiled coil domains, and the interaction with IE1 is important for Bin8 function. Ac16 also forms a complex with viral FP25 and cellular actin and associates with membranes via palmitoylation. These data suggested that this gene family encodes a multifunctional protein that accomplishes specific needs of group INPVs.     

Expression of <Emphasis Type="Italic">Trichoderma</Emphasis><Emphasis Type="Italic">viride</Emphasis> endoglucanase III in the larvae of silkworm, <Emphasis Type="Italic">Bombyx mori L.</Emphasis> and characteristic analysis of the recombinant protein

Endoglucanase is a part of cellulase which hydrolyzes cellulose into glucose. In this study, we cloned endoglucanase III (EG III) gene from Trichoderma viride strain AS 3.3711 using a PCR-based exon splicing method, and expressed EG III recombinant protein in both silkworm BmN cell line and silkworm larvae with an improved Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the chiA and v-cath genes of Bombyx mori nucleopolyhedrovirus (BmNPV). The result showed that around 45 kDa protein was visualized in BmN cells at 48 h after the second generation recombinant mBacmid/BmNPV/EG III baculovirus infection. The enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 8.0 and temperature 50°C, and increased 20.94 and 19.13% compared with that from blank mBacmid/BmNPV baculoviruses infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 9.0 and at temperature range from 40 to 60°C. It provided a possibility to generate transgenic silkworms expressing bio-active cellulase, which can catabolize dietary fibers more efficiently, and it might be of great significance for sericulture industry.     

<Emphasis Type="Italic">STM1</Emphasis>, a gene which encodes a guanine quadruplex binding protein,interacts with<Emphasis Type="Italic"> CDC13</Emphasis> in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The CDC13 gene encodes a protein that binds to the G-rich single-strand at yeast telomeres, and serves as a regulator of telomere replication. Cdc13 interacts with Est1 and DNA polymerase alpha, and cells carrying the temperature-sensitive allele cdc13-1 cannot complete telomere replication at the restrictive temperature and possess long telomeres. We attempted to isolate and characterize genes that interact with CDC13, in order to clarify the molecular mechanisms of telomere replication. A STM1 cDNA was isolated in a two-hybrid screen using CDC13 as a bait. The temperature-sensitive growth phenotype and the alteration in telomere size in cdc13-1 cells were corrected by introduction of the STM1 gene on a multicopy vector, but the extended G-rich single-strand overhangs which are also characteristic in the cdc13-1 mutant were not affected. Furthermore, we found that multiple copies of SGS1, a gene encoding a helicase that can unwind guanine quadruplexes, inhibited suppression of the cdc13-1 phenotype by STM1. We also demonstrate that a fusion protein consisting of the N-terminal region of Cdc13 and the C-terminal region of Stm1 (which shows similarity to the beta-subunit of the telomere binding complex in Oxytricha) could complement a cdc13 disruptant. Although STM1 itself is not essential for telomere replication, our findings suggest that STM1 genetically interacts with CDC13 to maintain telomere structure.     

Inhibition of apoptosis by <Emphasis Type="Italic">Heliothis virescens</Emphasis> ascovirus (HvAV-3e): characterization of <Emphasis Type="Italic">orf</Emphasis>28 with structural similarity to inhibitor of apoptosis proteins

Ascoviruses (AVs) induce a unique pathology in their insect host cells causing cleavage of the cells into virion-containing vesicles. The mechanism by which AVs induce vesicle formation is poorly understood. It is postulated that the virus initially induces apoptosis leading to cell fragmentation. The apoptotic bodies are however, rescued by the virus to form the vesicles. Here we show that Heliothis virescens AV (HvAV-3e) is able to inhibit chemically induced apoptosis from around 16 h after infection. Analysis of the genome of the virus indicated the presence of a putative inhibitor of apoptosis (orf28) gene that encodes a protein with an imperfect baculovirus inhibitor of apoptosis repeat (BIR) and a RING domain. Transiently expressed orf28 did not inhibit chemically induced apoptosis suggesting that the protein may not serve as an inhibitor of apoptosis. Nevertheless, RNA interference studies revealed that the gene is probably essential for virus pathology and replication.     

Analysis of synonymous codon usage bias in <Emphasis Type="Italic">helicase</Emphasis> gene from <Emphasis Type="Italic">Autographa californica</Emphasis> multiple nucleopolyhedrovirus

The helicase gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is not only involved in viral DNA replication, but also plays a role in viral host range. To identify the codon usage bias of helicase of AcMNPV, the codon usage bias of helicase was especially studies in AcMNPV and 41 reference strains of baculoviruses by calculating the codon adaptation index (CAI), effective number of codon (ENc), relative synonymous codon usage (RSCU), and other indices. The helicase of baculovirus is less biased (mean ENc?=?50.539?>?40; mean CAI?=?0.246). AcMNPV helicase has a strong bias toward the synonymous codons with G and C at the third codon position (GC3s?=?53.6%). The plot of GC3s against ENc values revealed that GC compositional constraints are the main factor that determines the codon usage bias of major of helicase. Several indicators supported that the codon usage pattern of helicase is mainly subject to mutation pressure. Analysis of variation in codon usage and amino acid composition indicated AcMNPV helicase shows the significant preference for one or more postulated codons for each amino acid. A cluster analysis based on RSCU values suggested that AcMNPV is evolutionarily closer to members of group I alphabaculovirus. Comparison of the codon usage pattern among E. coli, yeast, mouse, human and AcMNPV showed that yeast is a suitable expression system for AcMNPV helicase. AcMNPV helicase shows weak codon usage bias. This study may help in elucidating the functional mechanism of AcMNPV helicase and the evolution of baculovirus helicases.     

Sequencing and Characterization of Plasmid pUIBI-1 from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Serovar <Emphasis Type="Italic">entomocidus</Emphasis> LBIT-113

Plasmid pUIBI-1 from Bacillus thuringiensis svr. entomocidus was sequenced and its replication mechanism analyzed. Sequence analysis revealed that pUIBI-1 contains 4671 bp and a 32% GC content. Plasmid pUIBI-1 also includes at least seven putative open reading frames (ORFs) encoding for proteins ranging from 5 to 50 kDa. ORF-1 encodes for a putative 16-kDa Rep protein, which lacks homology with proteins of similar function. ORF2 encodes for a protein of 50 kDa and shows homology with Mob proteins of plasmids pLUB1000 from Lactobacillus hilgardii (32.2%) and pGI2 from B. thuringiensis (33.7%). Detection of single-stranded DNA (ssDNA) intermediates indicated that pUIBI-1 replicates by the rolling-circle replication mechanism, as demonstrated by S1 treatment and Southern hybridization under non-denaturing conditions.     

Shared molecular characteristics of successfully transformed mitochondrial genomes in <Emphasis Type="Italic">Chlamydomonas</Emphasis><Emphasis Type="Italic">reinhardtii</Emphasis>

Three types of respiratory deficient mitochondrial strains have been reported in Chlamydomonas reinhardtii: a deficiency due to (i) two base substitutions causing an amino acid change in the apocytochrome b (COB) gene (i.e., strain named dum-15), (ii) one base deletion in the COXI gene (dum-19), or (iii) a large deletion extending from the left terminus of the genome to somewhere in the COB gene (dum-1, -14, and -16). We found that these respiratory deficient strains of C. reinhardtii can be divided into two groups: strains that are constantly transformable and those could not be transformed in our experiments. All transformable mitochondrial strains were limited to the type that has a large deletion in the left arm of the genome. For these mitochondria, transformation was successful not only with purified intact mitochondrial genomes but also with DNA-constructs containing the compensating regions. In comparison, mitochondria of all the non-transformable strains have both of their genome termini intact, leading us to speculate that mitochondria lacking their left genome terminus have unstable genomes and might have a higher potential for recombination. Analysis of mitochondrial gene organization in the resulting respiratory active transformants was performed by DNA sequencing and restriction enzyme digestion. Such analysis showed that homologous recombination occurred at various regions between the mitochondrial genome and the artificial DNA-constructs. Further analysis by Southern hybridization showed that the wild-type genome rapidly replaces the respiratory deficient monomer and dimer mitochondrial genomes, while the E. coli vector region of the artificial DNA-construct likely does not remain in the mitochondria.     

Comparative Genomics of <Emphasis Type="Italic">Bifidobacterium</Emphasis>, <Emphasis Type="Italic">Lactobacillus</Emphasis> and Related Probiotic Genera

Six bacterial genera containing species commonly used as probiotics for human consumption or starter cultures for food fermentation were compared and contrasted, based on publicly available complete genome sequences. The analysis included 19 Bifidobacterium genomes, 21 Lactobacillus genomes, 4 Lactococcus and 3 Leuconostoc genomes, as well as a selection of Enterococcus (11) and Streptococcus (23) genomes. The latter two genera included genomes from probiotic or commensal as well as pathogenic organisms to investigate if their non-pathogenic members shared more genes with the other probiotic genomes than their pathogenic members. The pan- and core genome of each genus was defined. Pairwise BLASTP genome comparison was performed within and between genera. It turned out that pathogenic Streptococcus and Enterococcus shared more gene families than did the non-pathogenic genomes. In silico multilocus sequence typing was carried out for all genomes per genus, and the variable gene content of genomes was compared within the genera. Informative BLAST Atlases were constructed to visualize genomic variation within genera. The clusters of orthologous groups (COG) classes of all genes in the pan- and core genome of each genus were compared. In addition, it was investigated whether pathogenic genomes contain different COG classes compared to the probiotic or fermentative organisms, again comparing their pan- and core genomes. The obtained results were compared with published data from the literature. This study illustrates how over 80 genomes can be broadly compared using simple bioinformatic tools, leading to both confirmation of known information as well as novel observations.     

<Emphasis Type="Italic">tantalus</Emphasis>, a potential link between <Emphasis Type="Italic">Notch</Emphasis> signalling and chromatin-remodelling complexes

The tantalus (tan) gene encodes a protein that interacts specifically with the Polycomb/trithorax group protein Additional sex combs (ASX). Both loss-of-function and gain-of-function mutations in tan cause tissue-specific defects in the eyes, wing veins and bristles of adult flies. As these defects are also typical for components of the Notch (N) signalling pathway, we wished to determine if TAN interacts with this pathway. Through careful examination of ectopic tan phenotypes, we find that TAN specifically disrupts all three major processes associated with the N signalling pathway (boundary formation, lateral inhibition, and lineage decisions). Furthermore, ectopic tan expression abolishes expression of two N target genes, wingless (wg) and cut, at the dorsal-ventral boundary of the wing. An interaction between tan and N was also observed using a genetic assay that previously detected interactions between tan and Asx. The previously observed ability of TAN to move between the cytoplasm and nucleus, and to associate with DNA, provides a potential mechanism for TAN to respond to N signalling.Edited by P. Simpson     

Mitochondrial Genome of <Emphasis Type="Italic">Ciona savignyi</Emphasis> (Urochordata,Ascidiacea, Enterogona): Comparison of Gene Arrangement and tRNA Genes with <Emphasis Type="Italic">Halocynthia roretzi</Emphasis> Mitochondrial Genome

The complete nucleotide sequence of the urochordate Ciona savignyi (Ascidiacea, Enterogona) mitochondrial (mt) genome (14,737 bp) was determined. The Ciona mt genome does not encode a gene for ATP synthetase subunit 8 but encodes an additional tRNA^Gly gene (anticodon UCU), as is the case in another urochordate, Halocynthia roretzi (Ascidiacea, Pleurogona), mt genome. In addition, the Ciona mt genome encodes two tRNA^Met genes; anticodon CAT and anticodon TAT. The tRNA^Cys gene is thought to lack base pairs at the D-stem. Thus, the Ciona mt genome encodes 12 protein, 2 rRNA, and 24 tRNA genes. The gene arrangement of the Ciona mt genome differs greatly from those of any other metazoan mt genomes reported to date. Only three gene boundaries are shared between the Halocynthia and the Ciona mt genomes. Molecular phylogenetic analyses based on amino acid sequences of mt protein genes failed to demonstrate the monophyly of the chordates.     

Genome organization of <Emphasis Type="Italic">Treponema zioleckii</Emphasis>

Genome analysis of Treponema zioleckii proved that, in this bacterium, besides chromosomal DNA, a relatively small extrachromosomal DNA element is present. This element was shown to be a double-stranded circular plasmid DNA of approximately 7 kbp; it was designated as pKT. The plasmid was characterized by molecular and bioinformatic analysis. No pKT homologous DNA sequences were detected in other rumen Treponema strains. The overall G+C content of the pKT plasmid is approximately 56 %, which is higher than in other Treponema plasmids or genomes. The Rep module of the pKT plasmid consisting of the rep gene and the region of repeats was identified within a 1.6-kbp fragment. The putative rep gene encodes the replication protein belonging to the pfam04796 RepA_C family of proteins with the highest similarity (25 % within 249 amino acids) to the RepA protein from the green sulfur bacterium Prosthecochloris aestuarii.     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

Gene <Emphasis Type="Italic">RAD31</Emphasis> is identical to gene <Emphasis Type="Italic">MEC1</Emphasis> of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The earlier identified gene RAD31 was mapped on the right arm of chromosome II in the region of gene MEC1 localization. Epistatic analysis demonstrated that the rad31 mutation is an allele of the MEC1 gene, which allows further designation of the rad31 mutation as mec1-212. Mutation mec1-212, similar to deletion alleles of this gene, causes sensitivity to hydroxyurea, disturbs the check-point function, and suppresses UV-induced mutagenesis. However, this mutation significantly increases the frequency of spontaneous canavanine-resistance mutations induced by disturbances in correcting errors of DNA replication and repair, which distinguishes it from all identified alleles of gene MEC1.     

Nucleotide sequence and molecular characterization of the structural glycoprotein gp41 gene homologue of <Emphasis Type="Italic">Spodoptera litura</Emphasis> nucleopolyhedrosis virus (SpltNPV-I)

The structural glycoprotein gene gp41 homologue of Spodoptera litura nucleopolyhedrosis virus (SpltNPV-I *) was identified in the 4.0 kb EcoRI-L fragment of the viral genome. The nucleotide sequence of 2063 bp of this fragment revealed an open reading frame of 1014 nucleotides to encode a polypeptide of 337 amino acids. Analysis of nucleotide and deduced amino acid sequences of the putative ORF indicated its identity with gp41 protein of other baculoviruses sharing maximum homology with that of Spodoptera frugiperda nucleopolyhedrosis virus (SfNPV). The coding sequence was preceded by an AT-rich region containing the consensus baculoviral late promoter motif RTAAG. The putative SpltNPV gp41 ORF was abundantly expressed as a 37 kDa apoprotein in E. coli and as a 50 kDa glycoprotein in Sf9 cells. The recombinant protein expressed in insect cells was glycosylated (20%) and has GlcNAc as the terminal sugar. The gene is conserved among baculoviruses and places SpltNPV-I close to Spodoptera frugiperda and Spodoptera exigua NPVs in phylogenetic tree.Assigned GenBank accession no. for the nucleotide sequence data is AF445192.abbreviated as SlNPV in earlier publications and GenBank     

Characterization of the <Emphasis Type="Italic">Bm61</Emphasis> of the <Emphasis Type="Italic">Bombyx mori</Emphasis> Nucleopolyhedrovirus

orf61 (bm61) of Bombyx mori Nucleopolyhedrovirus (BmNPV) is a highly conserved baculovirus gene, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this study, we describe the characterization of bm61. Quantitative polymerase chain reaction (qPCR) and western blot analysis demonstrated that bm61 was expressed as a late gene. Immunofluorescence analysis by confocal microscopy showed that BM61 protein was localized on nuclear membrane and in intranuclear ring zone of infected cells. Structure localization of the BM61 in BV and ODV by western analysis demonstrated that BM61 was the protein of both BV and ODV. In addition, our data indicated that BM61 was a late structure protein localized in nucleus.